1. Field of the Invention
The present invention relates to a flow cytometry apparatus, and more particularly, concerns a flow cytometry apparatus for determining characteristics of cells or the like, which includes improved optics for obtaining uniform incoherent light excitation of the sample stream.
2. Description of the Prior Art
Flow cytometry apparatuses rely upon the flow of cells or other particles in a liquid flow stream in order to determine one or more characteristics of the cells under investigation. For instance, a liquid sample containing cells is directed through the flow cytometry apparatus in a rapidly moving liquid stream so that each cell passes serially, and substantially one at a time, through a sensing region. Cell volume may be determined by changes in electrical impedance as each cell passes through the sensing region. Similarly, if an incident beam of light is directed at the sensing region, the passing cells scatter such light as they pass therethrough. This scattered light has served as a function of cell shape, index of refraction, opacity, roughness and the like. Further, fluorescence emitted by labeled cells which have been excited as a result of passing through the excitation energy of the incident light beam is detectable for identification of specifically labeled cells. Not only is cell analysis performed on the flow cytometry apparatuses, but sorting of cells may also be achieved. Lasers have been used as the source of the incident beam of illumination in flow cytometry apparatuses, as well as sources of incoherent or non-collimated light, such as mercury or xenon arc lamps. Such apparatuses have been described in copending patent applications, Ser. No. 276,738, filed on June 24, 1981, in the U.S. Patent and Trademark Office and Ser. No. 482,346, filed in the U.S. Patent and Trademark Office on Apr. 5, 1983, both applications having a common assignee herewith, and also in U.S. Pat. No. 4,348,107. One flow cytometry apparatus known and sold as the FACS.TM. Analyzer, FACS Systems, Becton, Dickinson and Company, Sunnyvale, Calif., relies upon a mercury arc lamp as the excitation source of incoherent light for illuminating the stream of particles or cells flowing therethrough.
Excitation energy from a mercury or xenon arc lamp is typically bright and spectrally rich. However, as employed in a flow cytometry apparatus, the arc lamp may exhibit two undesirable characteristics. First, the brightness of the arc lamp may not be uniform over the used area of the source of the light. Hence, fluorescently labeled particles, flowing through the flow cytometry apparatus, at which such light is directed, may not be excited uniformly, and the resulting fluorescence emitted by these particles may not be uniform even though the particles themselves may be uniform in their ability to fluoresce. Second, the position of the arc itself, within the light source, may not be stable. As a result, additional fluctuations in the excitation intensity could be produced.
At the present time, there are two alternate techniques, known to those skilled in this art, to rectify the aforementioned undesirable traits associated with the ability of an arc lamp to produce uniform arc images in a flow cytometry apparatus. One technique has been to employ standard Kohler illumination techniques. When using these Kohler techniques, the image of the arc lamp is imaged into the pupil of the excitation objective lens. While this approach may produce uniform time-independent excitation, such excitation is typically spread over the entire field of view of the objective lens resulting in low excitation energy at the moving particle stream within the flow cytometry apparatus. A second technique utilized in this field involves the magnification of the arc image by a large factor followed by directing the enlarged image onto a small slit. Light passing through the slit is then imaged onto the flowing stream of particles by an objective lens. It has been found, however, that most of the light is lost before it reaches the particle flowing stream principally because the light does not pass effectively through the slit.
Accordingly, improvements are still being sought for flow cytometry apparatuses which rely upon an incoherent light source, such as mercury or xenon arc lamps, for producing light to illuminate the particles flowing in the liquid stream. Improvements are needed particularly in providing uniform light energy to the flowing particles so that uniform excitation thereof may be achieved especially when fluorescently labeled particles are being analyzed. It is to such improvement that the present invention is directed.